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Biochimica et Biophysica Acta, 547 (1979) 138--148 © Elsevier/North-Holland Biomedical Press

BBA 47689

CYTOCHROME FUNCTION IN THE CYCLIC ELECTRON TRANSPORT PATHWAY OF CHLOROPLASTS

R U D O L F E. SLOVACEK, DAVID CROWTHER and G E O F F R E Y HIND

Biology Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) (Received November 23rd, 1978)

Key words: Electron transport; Cytochrome b6; Cytochrome f; Photophosphorylation; Antimycin

Summary Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the s-bands of cytochrome b6 and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome be and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 pjVl 3-(3,4-dichlorophenyl)-l,l-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH4C1, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise c o m p o n e n t in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b6 and f by factors of ten or more. These observations indicate that cytochrome b6, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 ~zmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO2 fixation.

DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; CCCP, csLrbonylcyardde-rn-chlorophenylhydrazone; P-700, reaction center pigment for P h o t o s y s t e m I. Abbreviations:

139 Introduction Studies of intact chloroplasts and thylakoid membranes reconstituted with ferredoxin have shown that cyclic photophosphorylation driven by Photosystem I is inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6isopropyl-p-benzoquinone (DBMIB) [1,2] and by antimycin [2--4]. The observation that linear and cyclic electron flows are similarly sensitive to DBMIB and uncouplers prompted the suggestion [1,5] that these two processes share a c o m m o n coupling site between plastoquinone and cytochrome f. Antimycin, by contrast, is a specific inhibitor of the cyclic system; at antimycin concentrations which inhibit cyclic phosphorylation [2,3] or other energy-dependent parameters [6 ], linear electron flow is neither uncoupled nor inhibited [7]. The inhibition site for antimycin has thus been ascribed to a part of the cyclic pathway not shared with the linear pathway [3,4,7]. By analogy with mitochondria [8] and photosynthetic bacteria [9,10], this site is presumed to lie between the b- and c-type chloroplast cytochromes. Currently, only scant kinetic evidence has been presented for the participation of cytochromes bs and f in the chloroplast cyclic system [11], and there has been no evidence for an electrogenic loop between the chloroplast cytochromes, such as that observed in bacterial chromatophores [9,10] or with algae [31--33]. However, similar complex kinetics have been noted for the flash-induced 515 n m absorbance change in intact chloroplasts [34]. The results here form a preliminary kinetic study of cytochrome b6 and f turnovers and of the electrochromic effect in intact chloroplast preparations known to have cyclic activity. Materials and Methods Intact CO2-fixing chloroplasts were isolated from spinach as described previously [12]. Measurements at 18°C were performed with samples composed of chloroplasts in 'reaction buffer' consisting of 0.36 M sorbitol, 50 mM Tricine and 0.3 mM K2HPO4 adjusted to pH 8.1. Samples also contained 1200 units of catalase/ml to prevent H202 buildup during illumination [13]. The chlorophyll concentrations were 70 ug/ml for steady state and 50/~g/ml for flash-induced absorption change measurements. Steady-state spectra were obtained by the technique of Rapp and Hind [14] with the spectrophotometer in single beam mode. Flash-induced absorption changes with peaks at 518, 554 and 564 nm were kinetically resolved by the procedure of Dolan and Hind [11] with the following modifications: (i) elimination of the P-518 c o m p o n e n t from the signals at 554 and 564 nm was achieved by appropriate digital subtractions of the change at 518 nm rather than 531 nm (see Ref. 11 and text); (ii) flash intensity was doubled by illuminating the sample cuvette from opposing directions, at right angles to the measuring beam, with two EG and G FX201 xenon lamps; (iii) flash spectral composition was limited to greater than 650 nm by Kodak Wratten 70 filters and had a 4/~s pulse width at half peak height; (iv) flash frequency was 1.5 Hz. A single flash from both lamps resulted in at least 90% saturation of the 518 nm change. Resolution of the relaxation kinetics was achieved by non-linear least-squares regression analysis on a CDC

140

7600 computer. Calculated half-times fell within a standard error o f ±10% with the exception of those for c y t o c h r o m e f in Fig. 5a and c where the error was ±45%. Endogenous electron acceptors such as HCO~ were omitted from the reaction medium to prevent reoxidation of NADPH and p r o m o t e cyclic electron flow. Calculations, based on a photosynthetic unit size of 1/500 chlorophylls and a total NADP ÷ plus NADPH pool of 10 nmol/mg chlorophyll in the stroma [15], indicated that 20 flashes should completely reduce the NADP ÷ pool; consequently, 20--30 preilluminating flashes were given prior to acquisition of data. The p h o t o p h o s p h o r y l a t i o n of endogenous stromal ADP was measured as previously described [6]. Results Fig. 1 displays a light minus dark difference spectrum for intact chloroplasts subjected to weak continuous red illumination in the presence of monensin and NaC1. The absorbance changes associated with the ~-bands of cytochrome f (kmax = 554 nm) and c y t o c h r o m e b6 (kmax = 564 nm) are superimposed on a large positive signal with a peak at a b o u t 518 nm (P518). If 545 and 575 nm

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141 are assumed to be isosbests for the cytochrome responses [5,17], and the tail of the P-518 response is extrapolated within this region (dashed line) then the a-band difference spectrum shown in the inset can be obtained by subtraction. In this corrected spectrum cytochromes f and b6 are clearly responsible for the distinct peaks at 554 nm and 564 nm. The broad absorbances of plastocyanin [35], P-700 ÷ [23] and P-518 [11] in the a-band region may contribute to the net displacement observed in the spectrum. The above conclusion is reinforced by the flash excitation studies presented in Fig. 2. The absorbance spectrum 700 tLs after an excitation flash is predominantly that of the electrochromic effect (P-518) with peaks at 480 and 518 nm, as was also obtained by Vredenberg and Schapendonk [17] with intact chloroplasts. This spectrum also agrees well with that observed 320 tLs after flash illumination of Chlorella [18]. The cytochrome changes, at 700 us, are superimposed upon the tail of the P-518 signal as in the steady-state spectrum of Fig. 1. After 44 ms, the cytochrome changes have completely relaxed and the spectrum between 500 and 575 nm is essentially that of P-518 [11]. In order to remove the P-518 absorbance contribution from the a-band region, the 44 ms spectrum was normalized to match the 518 nm peak in the 700 us spectrum and then subtracted. The result, displayed in the inset of Fig. 2, shows that 700 tLs after the flash there is a substantial oxidation of cytochrome f together with a smaller apparent reduction of cytochrome b6. This finding agrees with earlier reports showing that cytochrome f is fully oxidized 700 tts after a flash [11,18] whereas cytochrome b6 is only partially reduced. The half-time for cytochrome b6 reduction has been estimated at 1.3 ms [11].

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142

From the above spectral observations was determined the number of repetitive absorbance transients, measured at 518 nm, required to strip the P-518 contribution from the absorbance changes in the a-band region. Values of 7.2% and 5.4% were used for the relative P-518 absorbance contribution at 554 and 564 nm, respectively, in subsequent curve-stripping procedures (Figs. 4 and 5). Fig. 3 documents the effect of flash frequency on the electrochromic change plotted as the relative amplitude at selected time intervals after the flash. The signal amplitude is relatively constant for repetitive flash frequencies between 0.5 and 2 Hz but at higher frequencies there is a linear decline in amplitude indicative of incomplete relaxation between flashes. This is consistent with the 500 ms dark recovery time for the electrochromic effect recently observed with intact chloroplasts [17]. The secondary rise in the 518 nm change (see Fig. 4b for example) shows a similar behavior with the minor exception that its peak height occurs at progressively shorter time intervals (as noted in brackets) after the flash. The dark relaxation of cytochromes f and b6 is essentially complete within 50 ms (cf. Fig. 2) therefore a flash frequency of 1.5 Hz was chosen to maximize both the cytochrome and electrochromic response in the following study. The oxidation kinetics of cytochrome b6 and the reduction kinetics of cytochrome f should both depend on the coupling state of the chloroplast if the cyclic pathway involves cytochrome b6 and utilizes the coupled step between plastoquinone and cytochrome f [1,5]. These expectations are in agreement with the flash kinetics for intact chloroplasts depicted in Fig. 4. When chloroplasts are examined without additions (Fig. 4a) the amplitudes of the cytochrome b6 and f responses to a flash are approximately equal and their dark

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relaxations nearly complete within 44 ms. Calculation gives a ratio of approximately 1.0 for cytochrome b6 to f heme turnover, based upon extinction coefficients of 17 and 22 mM -z • cm -z, respectively [16]. This suggests that, when the acceptor NADP ÷ is not regenerated due to lack of added electron acceptors, light